Plasmacytoid dendritic cells (pDCs) are critical innate immune cell that provide high levels of type I interferons (IFNs) in response to a variety of viruses. Type I IFN system plays a vital role in the early innate immune responses during virus infection. Until recently, the mechanism by which pDCs recognize viruses and elicit type I IFN secretion was not known. One of the major pathways of pathogen detection involves the recognition of evolutionary conserved patterns found on microbes via pattern recognition receptors. The Toll-like receptor (TLR) family of proteins are crucial in the activation of DCs and the generation of adaptive immunity following bacterial, parasitic and fungal infections. Yet, much less is known with regards to the role of TLRs in anti-viral immunity. In recent studies, we provided both in vitro and in vivo evidence for the requirement of TLR9 and TLR7 in type I IFN secretion from pDCs in response to a variety of viruses. Double stranded (ds) DNA viruses, herpes simplex viruses (HSV), are detected by TLR9 whereas single stranded (ss) RNA viruses, influenza and vesicular stomatitis virus (VSV), are recognized through TLR7. Further, recognition of both types of viruses through TLR7 and TLR9 required acidification of the endosomes. However, major gaps in our understanding of the pDCs exist in the following areas;(1) molecular mechanism of IFN induction upon viral infection and/or uptake, (2) cellular mechanism by which viruses are internalized into the lysosomal compartment, and (3) the role of pDCs in the context of in vivo innate and adaptive immunity to viruses. Thus, in this proposal, we describe systematic approaches to address these fundamental questions. Specifically, in Aim 1, we propose to identify the molecular mechanisms involved in the induction of type I IFN production upon recognition of live viruses in pDCs. Experiments proposed in Aim 2 will critically examine the endocytic pathways utilized by pDCs to internalize virions. Finally, in Aim 3, we propose to evaluate the in vivo role of pDCs in innate and adaptive immune responses to mucosal viral infections with influenza and HSV-2. Through basic understanding of the biology of pDCs, these studies will help to provide important foundation with which to design immunological interventions against viral diseases.